Through the life of an individual, bone places a continuing demand on the osteoprogenitor pathway for new bone cells to remodel and repair the matrix in response to mechanical and hormonal challenges. Recognizing the cellular and molecular stages of the osteoprogenitor lineage is a fundamental requirement for understanding diseases of bone, particularly when viewed as a consequence of a failure of the lineage to meet a challenge. An osteoprogenitor lineage model has been developed utilizing CollA1-GFP constructs in marrow stromal and neonatal calvarial cultures that allow the later steps of osteoblast differentiation to be viewed in real time and correlated with transgenic expression in intact bone. This proposal will focus on operationally defined stages of the lineage prior to preosteoblast development. Existing promoter LacZ transgenic mice will be tested for activation during the early phase of the lineage. Using the new marker transgenes plus improvement to those already in hand, the forward progression of the lineage will be blocked with agents that have major effects on bone in vivo to define the phenotypic consequences to bone nodule formation in the culture model. In addition, the controversial possibility that new osteoblasts can be generated from a pathway where existing osteoblasts dedifferentiate, proliferate and redifferentiate will be explored. The ultimate test of the cell culture model is the examination of mice with knockout mutations of genes likely to be essential to the normal progression of the lineage. Mice heterozygous for null mutations of genes so important to the multiple lineages that they are embryonic lethal will be obtained and crossed with GFP and LacZ transgenic mice to facilitate assessment of the osteoprogenitor lineage. The goal is to develop an approach for understanding the functional significance of a gene to the osteoprogenitor lineage that can be applied to the increasing number of knockout and ENU mutant mice with an unanticipated abnormality in bone mass. It is from this type of approach that candidate osteoporosis risk genes can be identified for subsequent verification studies in humans.